Crop production is affected by numerous abiotic environmental factors, with soil salinity and drought having the most detrimental effects. Approximately 70% of the genetic yield potential in major crops is lost due to abiotic stresses, and most major agricultural crops are susceptible to drought stress. Attempts to improve yield under stress conditions by plant breeding have been largely unsuccessful, primarily due to the multigenic origin of the adaptive responses (Barkla et al. 1999, Adv Exp Med Biol 464:77-89).
Considerable effort has focused on the identification of genetic factors that contribute to stress tolerance and on the genetic engineering of crop plants with increased stress tolerance. A number of genes have been identified whose expression or mis-expression is associated with drought tolerance, via a variety of different mechanisms. For instance, transformed tobacco that express maize NADP-malic enzyme display increased water conservation and gained more mass per water consumed than wild-type plants (Laporte et al. 2002, J Exp Bot 53:699-705). Significant research effort has focused on the plant hormone abscisic acid (ABA), which is involved in adaptation to various environmental stresses. Transgenic tobacco and transgenic Arabidopsis that overexpress the enzyme 9-cis-epoxycarotenoid dioxygenase (NCED), which is key to ABA biosynthesis, display improved drought tolerance (Qin et al. 2002, Plant Physiol 128:544-51; Iuchi et al. 2001, Plant J 27:325-33). Drought tolerance is often linked to salt tolerance, since both are associated with regulation of osmotic potential and turgor. Accordingly, transgenic plants that overexpress a vacuolar H+pump (H+-pyrophosphatase), which generates a proton gradient across the vacuolar membrane, display improved drought—and salt-stress, due to increased solute accumulation and water retention (Gaxiola et al. 2001, Proc Natl Acad Sci U S A 98:11444-9). Trehalose also contributes to osmoprotection against environmental stress. Potato plants the mis-express trehalose-6-phosphate synthase, a key enzyme for trehalose biosynthesis, show increased drought resistance (Yeo et al. 2000, Mol Cells 10:263-8).
Arabidopsis has served as a model system for the identification of genes that contribute to drought tolerance. For instance, researchers have identified numerous genes that are induced in response to water deprivation (e.g., Taji et al. 1999, Plant Cell Physiol 40:119-23; Ascenzi et al., 1997, Plant Mol Biol 34:629-41; Gosti et al. 1995, Mol Gen Genet 246:10-18; Koizumi et al. 1993 Gene 129:175-82) and cis-acting DNA sequences called ABA responsive elements (ABREs) that control ABA or stress responsive gene expression (Giraudat et al. 1994, Plant Mol. Biol. 26: 1557).
Several drought tolerant mutants of Arabidopsis have been identified. These include the recessive mutants abh1 (Hugouvieux et al. 2001, Cell 106: 477), era1-2 (Pei et al. 1998, Science 282: 286) and abi1-1Ri (Gosti et al. 1999, Plant Cell 11:1897-1909). The mutants era1-2 and abh1 were identified by screening for seedlings hypersensitive to ABA, while the mutant abi1-1Ri was isolated as an intragenic suppressor of the ABA insensitive mutant abi1-1. Dominant drought tolerant mutants were identified by over-expressing ABF3, ABF4 (Kang et al. 2002, Plant Cell 14:343-357) or DREB1A (Kasuga, 1999 Nature Biotech 17: 287). ABF3 and ABF4 encode basic-region leucine zipper (bZIP) DNA binding proteins that bind specifically ABREs. DREB1A encodes a protein with an EREBP/AP2 DNA binding domain that binds to the dehydration-responsive element (DRE) essential for dehydration responsive gene expression (Liu et al. 1998, Plant Cell 10: 1391). A dominant drought tolerant phenotype in tobacco was obtained by over-expressing the soybean BiP gene (Alvim et al. 2001, Plant Physiol 126, 1042).
Activation tagging in plants refers to a method of generating random mutations by insertion of a heterologous nucleic acid construct comprising regulatory sequences (e.g., an enhancer) into a plant genome. The regulatory sequences can act to enhance transcription of one or more native plant genes; accordingly, activation tagging is a fruitful method for generating gain-of-function, generally dominant mutants (see, e.g., Hayashi H et al., Science(1992) 258: 1350-1353; Weigel D, et al., Plant Physiology(2000) 122:1003-1013). The inserted construct provides a molecular tag for rapid identification of the native plant whose mis-expression causes the mutant phenotype. Activation tagging may also cause loss-of-function phenotypes. The insertion may result in disruption of a native plant gene, in which case the phenotype is generally recessive.
Activation tagging has been used in various species, including tobacco and Arabidopsis, to identify many different kinds of mutant phenotypes and the genes associated with these phenotypes (Wilson K et al., Plant Cell(1996) 8: 659-671, Schaffer R, et al., Cell(1998) 93: 1219-1229, Fridborg I et al., Plant Cell 11: 1019-1032, 1999; Kardailsky I et al., Science(1999) 286: 1962-1965; Christensen S et al., 9th International Conference on Arabidopsis Research. Univ. of Wisconsin-Madison, Jun. 24-28, 1998. Abstract 165).
We used activation tagging techniques to identify the association between the Arabidopsis DNA binding protein OBF1 and drought tolerance. OBF1 was first identified in a screen designed to characterize factors associated with proteins that bind to octopine synthase (ocs) promoter elements (Zhang et al. 1995, Plant Cell 7:2241-2252). The ocs elements are 20 bp DNA motifs that were originally found in the Agrobacterium osc promoter and were subsequently identified in the CaMV 35S promoter and in promoters of a class of plant stress-inducible genes such as glutathione S-transferase genes, which may be involved in responses to oxidative stress. Auxins and/or salicylic acid can induce genes with ocs promoter elements, possibly through changes in the redox potential. The Arabidopsis OBF4/OBF5 proteins are basic-region leucine zipper (bZIP) transcription factors that have been shown to interact with ocs elements. OBP1 was identified by its ability to bind OBF4 and OBF5, even in the absence of the target ocs elements.
The OPB1 protein sequence predicted by Zhang et al. is presented in Genbank entry GI 1212759 and is 13 amino acids shorter at the N-terminal end than the protein predicted by the Arabidopsis sequencing project (i.e., encoded by the same genomic sequence) and presented in GI 7630027.
OBP1 contains a highly conserved Dof domain, a zinc finger DNA-binding domain found in proteins from a number of plant species (Yanagisawa S, 1996, Trends Plant Sci. 1:213-214). OBP1 is constitutively expressed both in aerial portions of the plant and in roots; it is induced by auxin, salicylic acid, and cycloheximide (Kang and Singh, Plant J 21: 329-339, 2000).